This invention relates to an assay for the free portion of organic substances or analytes that are present in biological fluids in both a form bound to protein (or other binding species present in the fluid) and in a non-bound or free form, the free and bound forms being in equilibrium with one another.
For most physiologically active substances that can be found jointly in both a free form and a protein-bound form in biological fluids such as blood, it is currently thought that it is the concentration of the free form that may control the physiological responses associated with those substances and may therefore be more significant clinically than in the concentration of total substance which includes both free (or unbound) and protein-bound substance.
European Patent Specification No. 26103 describes an assay method of this kind. The method involves causing the analyte and a labelled derivative thereof to compete for reaction with a specific binder for the analyte. The amount of the labelled derivative of the analyte which has become bound to the specific binder is then measured, and the measurement used to determine the concentration of the free analyte in the biological fluid. The amounts of labelled derivative and specific binder used are insufficient to substantially affect the aforesaid equilibrium. Also, the labelled derivative of the analyte is chosen to be substantially non-reactive with the natural binders in the biological fluid.
The method of the aforesaid European Patent Specification No.26103 is concerned with a competition immunoassay in which the labelled species is a version of the analyte. By contrast, the present invention is concerned with an assay in which the labelled species is a version of the specific binder for the analyte. In this respect, the assay of the present invention is more closely related to one-site immunometric assays.
Three kinds of assay may be summarised as follows:
(i) In a typical competition immunoassay, the reagents are the analyte (antigen), a version of the analyte tagged with a label (e,g. a radioactive atom), and an added specific binder (antibody for the analyte). The amount of specific binder used is insufficient for reaction with all the analyte and the labelled version thereof. The analyte and the labelled version thereof compete for reaction with the specific binder, and become bound thereto in proportions which depend on the concentration of the analyte in the assay sample. Then the bound portion of the labelled version of the analyte is separated from the non-bound portion, for which purpose the specific binder may be provided for example as a coating on solid particles or on the walls of the assay tube. The amount of labelled version of the analyte which is bound to the specific binder is inversely proportional to the concentration of the analyte in the assay sample.
(ii) In a typical one-site immunometric assay, the reagents are the analyte (antigen), a matrix-bound version of the analyte, and a specific binder (antibody) for the analyte. The amount of specific binder used is at least sufficient for reaction with all the analyte in the assay sample. In contrast to i), it is the specific binder which is tagged with a label (e.g. a radioactive atom). The analyte of the assay sample is reacted with the labelled specific binder. Then a sufficient amount of the matrix-bound analyte is added to react with excess labelled specific binder. Then the portion of the labelled specific binder bound to the matrix-bound analyte is separated from the portion not so bound. The amount of the specific binder which is bound to the matrix-bound analyte is inversely proportional to the concentration of the analyte in the assay sample. Immunometric assays are the subject of a review in British Medical Bulletin, 1974, pages 44 to 49.
(iii) Immunoassays have been described which are intermediate between (i) and (ii). See Journal of Steriod Biochemistry, 11, 1597-1600; and FEBS Letters, 96, 2, (Dec. 1978), 298-300. The reagents are, as in immunometric assay (ii), the analyte (antigen), a matrix-bound version of the analyte, and a labelled specific binder (antibody) for the analyte. The method, as in immunoassay (i), involves causing the analyte in the assay sample and the matrix-bound version thereof to compete for reaction with the labelled specific binder and to become bound thereto in proportions which depend on the concentration of the total analyte in the assay sample.
It is recognised that assays in which the specific binder is labelled are different from, and in some circumstances better than, assays in which a version of the analyte is labelled. For example, it may in some circumstances be easier to label an antibody than an antigen. Practical development of assays in which the specific binder is labelled have been delayed by the difficulty of providing labelled antibodies of sufficient purity. With the development of monoclonal antibodies, this difficulty is being overcome, and it is expected that such assays will in future achieve increasing importance.
This invention provides an assay in class (iii) which uses a labelled specific binder for the analyte, which assay is adapted to measure the free portion of an analyte present in a biological fluid in equilibrium with a portion of the analyte bound to one or more natural binders.